Immunocontraceptive Efficacy of Native Porcine Zona Pellucida (pZP) Treatment of Nevada's Virginia Range Free-Roaming Horse Population.

In North America, range constraints due to burgeoning development increasingly encroach on wild horse habitat and necessitate effective but humane reproductive management. The largest free-roaming wild horse fertility control program by population (>3500) and territory size (≈300,000 acres) is located within Nevada's Virginia Range. Data from a field study investigated porcine zona pellucida (pZP) immunocontraception via remote dart delivery to mares in this population. Analyses aimed to measure efficacy by treatment effects on annual birth rates and population demographics and to evaluate treatment frequency and season against these variables. Analyses included mares' monthly data (January 2019-December 2022; 48 months), characterized by cumulative vaccination numbers subset into four classifications considering the vaccine as having no loss of efficacy or a loss within a 6-, 12-, and 18-month period post vaccination; from foaling data, the likelihood of being in foal and of conceiving in that month; and from age, as mature or immature (<1 years-old). A downward foaling rate and trend in the numbers of mature mares, descriptively presented at monthly intervals, showed markedly declining annual seasonal breeding peaks, with no observed change in foaling season or duration. Within four years, population coverage surpassed 70% and was associated with a 58% reduction in foaling, with only a 10% conception rate. Vaccinated mares increased proportionally: assuming a 12-month decay rate, the system reached stability at an average ≈1.0 vaccination/mare/year, providing a robust recommendation for treatment frequency contributing to best management practices.

Effects of an anti-gonadoliberin releasing hormone vaccine on testicular, epididymal and spermatogenic development in the horse.

The effects of the GnRH vaccine Improvac® on testicular and epididymal morphometrics, histology and spermatogenesis were measured in 19 young (15-20 months) colts randomly assigned to one control (saline, castration at 57 days, n = 6) or either of two GnRH vaccine-treatment groups, T-57 (castration at 57 days, n = 7) or T-100 (castration at 100 days, n = 6), respectively. All were immunized on Day 0 with a single booster on Day 28. Excised testes and epididymides were weighed and processed for histology to measure tubule, epithelial and muscle dimensions, the ratio of interstitial tissue to seminiferous tubules and determine the stage of spermatogenesis. Testis volume, unchanged within controls, decreased in T-57 and T-100 groups by 50% and 70%, respectively. Treated colts' testes were significantly lighter than controls (64% relative difference); however, epididymal mass showed no significant differences between groups. Proportionally less seminiferous tubule relative to interstitial tissue was observed in both treatment groups (5%) versus controls (22%) with a mean tubule size 28% smaller than controls. Controls exhibited a high proportion of seminiferous tubules with advanced stages of spermatogenesis, whereas treated colts showed a high proportion of tubules in the early stages of spermatogenesis. In conclusion, immunization against GnRH in prepubertal colts was effective at reducing the development of their intra-scrotal reproductive organs and preventing normal spermatogenesis. GnRH vaccination of young colts effectively and consistently reduced testis mass, tubule size and relative proportion of seminiferous tubule tissue while retarding spermatogenesis. The epididymis showed changes with a smaller tubule diameter, lower epithelial height and thicker muscle layer recorded in treated compared to control colts.

Vaccination against GnRH as a prelude to surgical castration of horses.

BACKGROUND: Surgical castration is one of the most frequently performed surgeries in horses and is associated with various post-surgical complications ranging in severity. OBJECTIVES: We investigated the effects of administering an anti-GnRH vaccine to colts for reduction of testis size on the incidence of complications associated with their subsequent surgical castration. STUDY DESIGN: Randomised open controlled trial. METHODS: Nineteen colts were randomly assigned to one of the three groups. Treatment Groups V1-57 and V2-100 received two treatments of Improvac® (Zoetis, South Africa) before their castration in the field on either Day 57 or Day 100 following first treatment, respectively. Controls similarly received placebo treatments followed by castration on Day 57. Serial measurements of testicular dimensions and serum samples for measuring serum testosterone concentrations (STCs) and anti-GnRH antibody titres (ABTs) were obtained pre- and post-vaccination and on the day of castration. Clinical data recorded for 10 days determined post-surgical complications. RESULTS: All vaccinated colts showed a baseline STC concentration at castration with a strong ABT response. Mean testicular volume of Groups V1-57 and V2-100 reduced by 49.7% and 30.8%, respectively, on Day 57 and this further reduced in Group V2-100 by 63.9% at castration on Day 100. Testis size was significantly correlated with post-surgical preputial (P = 0.001) and scrotal (P = 0.025) swelling. MAIN LIMITATIONS: A study population of young light horse breed colts and a relatively small sample size limited this pilot study. CONCLUSIONS: Administering two doses of Improvac® in colts 28 days apart prior to castration effectively reduced their testicular volumes with associated significant post-surgical improvement in both the incidence and degree of local swelling. These results may inform mitigation of potential post-surgical complications associated with castration in the field.

Distribution of uterine histological changes in aged captive cheetahs (Acinonyx jubatus).

The histological effect on the felid uterus of sterilization, via ovariectomy or salpingectomy, is currently unknown. To investigate the association of ovariectomy or salpingectomy with uterine health, it is first necessary to establish if changes are distributed evenly throughout the uterus. Both laparoscopic ovariectomy and salpingectomy with concurrent sampling of the tip of the uterine horn are possible in the cheetah. Currently accepted practice for histopathological screening of the uterus utilizes four biopsy samples. It is not known whether this method accurately reflects the status of the entire uterus. In this study we histologically examined the uteri of six older cheetahs (one 7-year-old and five 10-10.5-year-old animals) via 21 tissue samples (three samples from seven different anatomical regions) per cheetah to determine overall uterine health. Although no defined lesions were detected, mild endometrial gland dilation, assumed to be of no functional consequence, was observed in multiple samples. The odds of observing this dilation was lowest in the uterine body and progressively increased in a cranial direction, being significantly higher at the tip of the uterine horns (OR = 11.5; 95% CI, 2.0-65.1; p = 0.006). This supported the reliability of sampling the tip of the uterine horn to screen for endometrial gland dilation.

Direct culture-independent sequence typing of Taylorella equigenitalis obtained from genital swabs and frozen semen samples from South African horses.

We report herein the use of crude extracts obtained from samples of Taylorella equigenitalis-infected horses for the purpose of multi-locus sequence typing (MLST). Samples (n = 36) were collected from horses in South Africa from 1996 to 2017: 34 from genital swabs (stored at -20°C for 2-3 y) and 2 from cryopreserved raw semen aliquots (stored at -70°C for 18 y) prior to assay. The MLST assay showed a single sequence type (ST), designated ST4, that supported a point introduction and thus a common source for the South African outbreak of contagious equine metritis.

Researching immunocontraceptive vaccines with mares (Equus caballus) as both a target and model for African elephant (Loxodonta africana) cows: A review.

A sequence of studies is reviewed that reported the domestic horse (Equus caballus) mare as an appropriate and accessible research platform for recording clinical and laboratory data post-immunisation with anti- GnRH and -zona pellucida (ZP) immunocontraceptive vaccines. Experience with a native porcine ZP (pZP) vaccine in African elephant (Loxodonta africana) cows highlighted needs for improving vaccine formulations and more clearly defining associated ovarian effects and safety profiles. Initially, the efficacy, reversibility and safety of the GnRH vaccine Improvac® in mares was demonstrated using reproductive tract ultrasonography and concurrently measuring serum antibody titres and progesterone concentrations. Results informed the study design and minimally invasive monitoring of post-treatment ovarian steroid responses of this vaccine in free-ranging African elephant cows. A subsequent sequence of studies reported reversible contraceptive and immunological efficacy in pony mares immunised with pZP formulated with Freund's adjuvants. By comparison, mares treated with a recombinant ZP3 and ZP4 (reZP) vaccine showed disappointing responses. Unexpectedly, most pZP-treated mares showed ovarian inactivity. In attempting to understand this response, results showed the involvement of cytotoxic (CD8+) T-cells negatively correlated to serum ovarian steroid and anti-Müllerian hormone (AMH) levels. Of concern was the prevalence of injection-site lesions ascribable to Freund's adjuvants. Following this, mares treated with both pZP and a novel reZP vaccine formulated with non-Freund's adjuvants showed comparable immunological responses and ovarian inactivity, notably without adverse treatment reactions. In addition, measuring AMH showed promise for monitoring ovarian function in anti-ZP-treated animals.

Failure to detect equid herpesvirus types 1 and 4 DNA in placentae and healthy new-born Thoroughbred foals.

Equid herpesvirus type 1 is primarily a respiratory tract virus associated with poor athletic performance that can also cause late gestation abortion, neonatal foal death and encephalomyelopathy. Horizontal transmission is well described, whereas evidence of vertical transmission of equid herpesvirus type 1 associated with the birth of a healthy foal has not been demonstrated. This study sampled a population of Thoroughbred mares (n = 71), and their healthy neonatal foals and foetal membranes, to test for the presence of both equid herpesvirus types 1 and 4 using a quantitative polymerase chain reaction assay. Foetal membrane swabs and tissue samples were taken immediately post-partum, and venous blood samples and nasal swabs were obtained from both mare and foal 8 h after birth. Neither equid herpesvirus type 1 nor equid herpesvirus type 4 nucleic acid was detected in any sample, and it was concluded that there was no active shedding of equid herpesvirus types 1 and 4 at the time of sampling. Consequently, no evidence of vertical transmission of these viruses could be found on this stud farm during the sampling period.

Serum antibody immunoreactivity and safety of native porcine and recombinant zona pellucida vaccines formulated with a non-Freund's adjuvant in horses.

Commercial and regulatory limitations associated with native porcine zona pellucida (pZP) vaccines formulated with Freund's adjuvants may be overcome by developing effective recombinant ZP vaccines (reZP) and identifying alternative adjuvant formulations. A two-part preparatory study used 15 geldings and identified potentially effective alternative adjuvant formulations based on anti-pZP antibody response following treatment with pZP formulated with Addavax (AddaVax ™, Invivogen), Quil A (Quil-A® Adjuvant, Invivogen), Quil A and Poly (I:C) (HMW VacciGrade™, Invivogen), Pet Gel A (Montanide™ Pet Gel A, Seppic) and Pet Gel A and Poly (I:C). Injection site reactions, rectal temperature and respiratory and heart rates were also monitored for three days post-treatment. Suitable anti-pZP antibody titres were seen in response to Pet Gel A and Pet Gel A and Poly (I:C). Subsequently in 31 mares, following administration of pZP, reZP and a combination of pZP and reZP proteins prepared in Pet Gel A and Poly (I:C), both serum anti-pZP and -reZP antibody responses were monitored. In addition, safety was assessed for up to seven days post-treatment by inspection and palpation of gluteal intramuscular injection sites and measurement of rectal temperature. The measured antibody titres in all treatment groups differed significantly to an adjuvant control group (P < 0.001). Temporal changes in both anti-pZP and -reZP antibody titres in all ZP treatment groups were similar to patterns reported previously in various species vaccinated with pZP formulated with Freund's adjuvants. There were no differences in anti-pZP antibody titres between the pZP and reZP treated groups (P > 0.05). Side effects were mild and transient in nature. This represents the first application of a reZP vaccine formulated with non-Freund's adjuvants evoking a similar antibody titre response to native pZP vaccination in mares.

Ovarian function following immunocontraceptive vaccination of mares using native porcine and recombinant zona pellucida vaccines formulated with a non-Freund's adjuvant and anti-GnRH vaccines.

An important determinant in the selection of any contraceptive agent is the impact on ovarian function, both in the short and longer term. In this study, ovarian activity was monitored in mares immunised with one of the following vaccine formulations; native porcine zona pellucida (pZP), recombinant zona pellucida proteins ZP3 and ZP4 (reZP), pZP and reZP combined or a commercially available anti-GnRH vaccine. The ZP antigens were prepared in an adjuvant formulation consisting of 6% polymeric adjuvant (Montanide™ PetGel A, Seppic, France) and 500 µg polyinosinic-polycytidylic acid - TLR3-agonist (Poly(I:C) HMW VacciGrade™, Invivogen, USA). A vehicle-only control group was administered the adjuvant formulation without antigen. Ovarian activity was monitored using clinical observations (transrectal palpation and ultrasonography of the reproductive tract) in addition to blood sampling for serum progesterone and anti-Müllerian hormone (AMH) concentrations while employing a low sampling frequency. Treatments and measurements were initiated in December (southern hemisphere summer) and subsequent data collection was performed in January, February, March and May. Both reZP and anti-GnRH vaccination were associated with clinically evident ovarian suppression in the short term. Ovarian activity in mares administered a reZP or anti-GnRH vaccine was significantly different to adjuvant control and pZP treated mares. Serum AMH concentrations were different between pZP and anti-GnRH treated mares 3.5 months after the final vaccination. Serum AMH concentrations were significantly correlated with mare age, serum progesterone and ovarian volume.

Ovarian dysfunction associated with zona pellucida-based immunocontraceptive vaccines.

Despite more than 40 years of research into zona pellucida (ZP)-based vaccines, relatively little is known about their mechanism of action. Early research demonstrated precipitation of ZP glycoproteins by antiovarian antiserum, rendering oocytes resistant to sperm binding in vitro. Subsequent work showed significantly decreased fertilization rates following passive immunization, sparking interest in anti-ZP immunocontraception for human and animal use. The primary mechanism of action of ZP vaccines is generally considered to be an antibody-mediated interference with sperm-oocyte binding and/or fertilization. However, this mechanism of action excludes the potential for ovarian dysfunction associated with anti-ZP treatment in some species. A review of relevant literature in pertinent model, domestic and wildlife species reveals a variety of previous and current hypotheses for ovarian effects following ZP-based immunization. Ovarian dysfunction has been suggested to be a species-specific response. In addition, cytotoxic T-lymphocytes and the use of Freund's adjuvants have been suggested to play a role. Finally, the type and extent of glycosylation of ZP antigens have been proposed to influence ovarian effects. The validity of these hypotheses is re-examined in the light of current knowledge. Further investigation of ovarian function in species believed to be resistant to the ovarian effects of anti-ZP vaccines is warranted. To this end, anti-Müllerian hormone may provide a novel tool for the assessment of ovarian function during ZP-based immunocontraception, particularly in wildlife species not amenable to frequent clinical examination.

Missense Mutation in the Ligand-Binding Domain of the Horse Androgen Receptor Gene in a Thoroughbred Family with Inherited 64,XY (SRY+) Disorder of Sex Development.

Disorders of sex development (DSD) have long been documented in domestic animal species including horses. However, there is only a single report of an androgen receptor (AR) mutation causative of such a DSD syndrome in a horse pedigree. Here, we present a new familial AR mutation in horses. A missense mutation (c.2042G>C) at AR exon 4 explains the segregation of the DSD in a Thoroughbred horse pedigree. The mutation, expected to affect the ligand-binding domain of the AR protein, led to complete androgen insensitivity of 64,XY SRY+, testicular DSD individuals. Additionally, the design of a PCR-RFLP technique provided an accurate molecular test for the identification of horses carrying the mutation.

Draft Genome Sequence of Taylorella equigenitalis Strain ERC_G2224 Isolated from the Semen of a Lipizzaner Stallion in South Africa.

Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We report here the genome sequence of T. equigenitalis strain ERC_G2224, isolated in 2015 from a semen sample collected in 1996 from a Lipizzaner stallion in South Africa.

Single-Incision Laparoscopic Sterilization of the Cheetah (Acinonyx jubatus).

OBJECTIVE: To describe laparoscopic ovariectomy and salpingectomy in the cheetah (Acinonyx jubatus) using single-incision laparoscopic surgery (SILS). STUDY DESIGN: Prospective cohort. ANIMALS: Female cheetahs (Acinonyx jubatus) (n = 21). METHODS: Cheetahs were randomly divided to receive either ovariectomy (n = 11) or salpingectomy (n = 10). The use and complications of a SILS port was evaluated in all of cheetahs. Surgery duration and insufflation volumes of carbon dioxide (CO2 ) were recorded and compared across procedures. RESULTS: Laparoscopic ovariectomy and salpingectomy were performed without complications using a SILS port. The poorly-developed mesosalpinx and ovarian bursa facilitated access to the uterine tube for salpingectomy in the cheetah. The median surgery duration for ovariectomy was 24 minutes (interquartile range 3) and for salpingectomy was 19.5 minutes (interquartile range 3) (P = .005). The median volume of CO2 used for ovariectomy was 11.25 L (interquartile range 3.08) and for salpingectomy was 4.90 L (interquartile range 2.52), (P = .001) CONCLUSIONS: Laparoscopic ovariectomy and salpingectomy can be performed in the cheetah using SILS without perioperative complications. Salpingectomy is faster than ovariectomy and requires less total CO2 for insufflation.

Laparoscopic sterilization of the African Lioness (Panthera leo).

OBJECTIVE: To describe laparoscopic ovariectomy and salpingectomy in the African lioness (Panthera leo). STUDY DESIGN: Descriptive clinical study. ANIMALS: Female lions (n = 16). METHODS: Lionesses were randomly divided into 2 groups: O = ovariectomy (n = 8) and S = salpingectomy (n = 8) for laparoscopic surgery. Two Veress needle placement techniques were used. RESULTS: Laparoscopic ovariectomy and salpingectomy were performed without complications. The poorly developed mesosalpinx and ovarian bursa rendered the uterine tube more accessible for salpingectomy compared to the dog making the procedure easier in the lioness. Similarly, salpingectomy is a relatively easier procedure compared with ovariectomy especially when the modified Hasson technique for Veress needle placement is used. CONCLUSIONS: Laparoscopic ovariectomy and salpingectomy can be safely performed in the African lioness.

Non-invasive assessment of the reproductive cycle in free-ranging female African elephants (Loxodonta africana) treated with a gonadotropin-releasing hormone (GnRH) vaccine for inducing anoestrus.

BACKGROUND: In southern Africa, various options to manage elephant populations are being considered. Immunocontraception is considered to be the most ethically acceptable and logistically feasible method for control of smaller and confined populations. In this regard, the use of gonadotropin-releasing hormone (GnRH) vaccine has not been investigated in female elephants, although it has been reported to be safe and effective in several domestic and wildlife species. The aims of this study were to monitor the oestrous cycles of free-ranging African elephant cows using faecal progestagen metabolites and to evaluate the efficacy of a GnRH vaccine to induce anoestrus in treated cows. METHODS: Between May 2009-June 2010, luteal activity of 12 elephant cows was monitored non-invasively using an enzyme immunoassay detecting faecal 5alpha-reduced pregnanes (faecal progestagen metabolites, FPM) on a private game reserve in South Africa. No bulls of breeding age were present on the reserve prior to and for the duration of the study. After a 3-month control period, 8 randomly-selected females were treated twice with 600 micrograms of GnRH vaccine (Improvac®, Pfizer Animal Health, Sandton, South Africa) 5-7 weeks apart. Four of these females had been treated previously with the porcine zona pellucida (pZP) vaccine for four years (2004-2007). RESULTS: All 12 monitored females (8 treated and 4 controls) showed signs of luteal activity as evidenced by FPM concentrations exceeding individual baseline values more than once. A total of 16 oestrous cycles could be identified in 8 cows with four of these within the 13 to 17 weeks range previously reported for captive African elephants. According to the FPM concentrations the GnRH vaccine was unable to induce anoestrus in the treated cows. Overall FPM levels in samples collected during the wet season (mean 4.03 micrograms/gram dry faeces) were significantly higher (P<0.002) than the dry season (mean 2.59 micrograms/gram dry faeces). CONCLUSIONS: The GnRH vaccination protocol failed to induce anoestrus in the treated female elephants. These results indicate that irregular oestrous cycles occur amongst free-ranging elephants and are not restricted to elephants in captivity. The relationship between ecological conditions and endocrine activity were confirmed. Free-ranging female elephants were observed to not cycle continuously throughout the year in the absence of adult bulls.

Ultrasonographic and laparoscopic evaluation of the reproductive tract of the captive female African lion (Panthera leo).

The use of transabdominal ultrasonography to assess the oestrous cycle has not been previously described in the African lion (Panthera leo). Twelve sexually mature lionesses and five female cubs had their reproductive organs assessed by transabdominal ultrasound. Ovarian findings were compared to laparoscopic findings while performing laparoscopic ovariectomy or salpingectomy. Vaginal cytology was performed and serum progesterone levels were determined. By combining all data the oestrous cycle stage of each lion was determined. One lion was far pregnant and was not operated on. In adults a uterine body could be seen ultrasonographically in 67% of lions while mural structures could be distinguished in 44% of lions. Five uterine horns could be seen in 3 lions. In 12 adults 10 ovaries were found of which eight had discernable follicles or luteal structures. During laparoscopy 12 active ovaries were seen with luteal structures seen in 11 ovaries and follicles in 2 ovaries. Using laparoscopy as the gold standard, ultrasonography had a sensitivity of 66% and specificity of 83% to detect ovarian reproductive activity. Two uterine cysts and a cluster of periovarian cysts were seen in three different lions. Three lions were pregnant, two were in oestrus, three in a luteal phase (dioestrus), and four were in anoestrus. Transabdominal ultrasound in combination with serum progesterone levels and vaginal cytology can be used to assess ovarian cyclical activity with reasonable accuracy in captive bred lions.